Coffea Pseudozanguebariae Research Articles

LC-MS methods combination for identification and quantification of trans-sinapoylquinic acid regioisomers in green coffee.

The present study aims to both identify and quantify trans-sinapoylquinic acid (SiQA) regioisomers in green coffee by combined UHPLC-ESI-QqTOF-MS/MS and UHPLC-ESI-QqQ-MS/MS methods. Among the various mono-acyl chlorogenic acids found in green coffee, SiQA regioisomers are the least studied despite having been indicated as unique phytochemical markers of Coffea canephora (known as Robusta). The lack of commercially available authentic standards has been bypassed by resorting to the advantages offered by high-resolution LC-MS as far as the identification is concerned. SiQA regioisomers have been identified in several samples of Robusta and Coffea arabica (known as Arabica) commercial lots from different geographical origin and, for the first time, in different samples of coffee wild species (Coffea liberica and Coffea pseudozanguebariae). Quantification (total SiQA ranging from 3 to 5mg/100 g) let to reconsider these chlorogenic acids as unique phytochemical markers of Robusta being present in the same quantity and distribution in C.liberica as well. Gardeniae Fructus samples (fruits of Gardenia jasminoides) have additionally been characterized as this matrix is recognized as one of the few naturally occurring SiQA sources. The SiQA regioisomer content (total SiQA about 80 mg/100 mg) fully supports the proposal to use this matrix as a surrogate standard for further studies.

Inheritance and relationship between key agronomic and quality traits in an interspecific cross between Coffea pseudozanguebariae Bridson and C. canephora Pierre

The inheritance and relationships between four traits of agronomic and quality interest—fructification time, caffeine, and heteroside contents and 100-bean weight—were analyzed in the first backcross hybrids derived from an interspecific cross between Coffea pseudozanguebariae and Coffea canephora. We showed that short vs. long fructification time was governed by one major gene with two co-dominant alleles ft1 and ft2. Absence vs. presence of both caffeine and heteroside was also controlled by one major gene. The allele responsible for the presence of caffeine (caf2) dominated over the absence one (caf1) whereas both alleles controlling heteroside, het1 and het2, were co-dominant. The fructification time and the heteroside content were additive while the caffeine content seemed multiplicative. The 100-bean weight was additive and under a polygenic control. The two genes ft and caf were linked, separated by 30.8 cM, and were independent from the het gene. The relationships between the four traits were not strong enough, except between 100-bean weight and fructification time (r = 0.43) or caffeine content (r = 0.41). Recombination occurred between the genes controlling the four traits suggesting that new introgressed Robusta varieties, characterized by short, medium, or long fructification time depending on demand, bigger seeds with low or no caffeine content, and being heteroside-free, could be produced.

Isolation of C-glycosyl Xanthones from Coffea pseudozanguebariae and Their Location

The biochemical composition of leaves from Coffea pseudozanguebariae, a wild caffeine-free coffee species, was determined. Two phenolic compounds were extracted from leaves, separated and characterized. Their structures were elucidated by mass spectrometry, and 1D and 2D NMR spectroscopy and were shown to be mangiferin (1) and isomangiferin (2), which were the main polyphenol products. Multiphoton fluorescence imaging was performed to visualize polyphenol distribution in leaf cross sections. Consistent biochemical analysis cell imaging techniques on leaves revealed yellow fluorescence in the epidermis and parenchyma cells corresponding to xanthone compounds.

First report on mangiferin (C-glucosyl-xanthone) isolated from leaves of a wild coffee plant, Coffea pseudozanguebariae (Rubiaceae)

Bean biochemical composition has been extensively analyzed in Coffea species because of its impact on coffee beverage quality. C. pseudozanguebariae appeared as an interesting wild species because of its low caffeine and chlorogenic acid content, compounds involved in coffee bitterness. Extending the study to its leaves revealed the presence of two unknown phenolic compounds. Isolation, then identification by MS and NMR analysis proved that these compounds are two C-glucosyl-xanthones: mangiferin and isomangiferin. HPLC analyses indicated very high mangiferin content in young leaves. This class of compounds is described for the first time not only in coffee plants but also in the Rubiaceae family. The interest of mangiferin identification in the Coffea genus is discussed.

Isolation and genetic mapping of a Coffea canephora phenylalanine ammonia-lyase gene (CcPAL1) and its involvement in the accumulation of caffeoyl quinic acids

Biosynthesis of caffeoylquinic acids occurs via the phenylpropanoid pathway in which the phenylalanine ammonia-lyase (PAL) acts as a key-control enzyme. A full-length cDNA (pF6), corresponding to a PAL gene (CcPAL1), was isolated by screening a Coffea canephora fruit cDNA library and its corresponding genomic sequence was characterized. Amplification of total DNA from seven Coffea species revealed differences in intronic length. This interspecific polymorphism was used to locate the gene on a genetic map established for a backcross progeny between Coffea pseudozanguebariae and C. dewevrei. The CcPAL1 gene was found on the same linkage group, but genetically independent, as a caffeoyl-coenzyme A-O-methyltransferase gene, another gene intervening in the phenylpropanoid pathway. In the same backcross, a lower caffeoylquinic acid content was observed in seeds harvested from plants harbouring the C. pseudozanguebariae CcPAL1 allele. Involvement of the CcPAL1 allelic form in the differential accumulation of caffeoylquinic acids in coffee green beans is then discussed.

Base Composition of Coffea AFLP Sequences and Their Conservation Within the Genus

Amplified fragment length polymorphism (AFLP) is often used for genetic mapping and diversity analysis, but very little information is currently available on their sequence characteristics. Species-specific sequences were analyzed from a single Coffea genome (Coffea pseudozanguebariae) associated with clustered or nonclustered AFLP loci of known genetic position. Compared with the expressed sequence tag (EST) sequence composition, their AT content exhibited a bimodal distribution with AT-poor sequences corresponding mainly to putative coding sequences. AT-rich sequences, apart from the EST distribution, were usually clustered on the genetic map and might correspond to noncoding sequences. Conversion of these AFLP markers into sequence-characterized amplified region (SCAR) anchor markers allowed us to assess sequence conservation within Coffea species with respect to species relatedness.

SSR cross-amplification and variation within coffee trees (Coffeaspp.)

Primer sets were developed from 85 Coffea arabica sequences in addition to 25 already published primer sets. They were subsequently used for amplification in six African Coffea species: Coffea canephora (CAN), Coffea eugenioides (EUG), Coffea heterocalyx (HET), Coffea liberica (LIB), Coffea sp. Moloundou (MOL) and Coffea pseudozanguebariae (PSE). The amplification percentages for these 110 primer pairs ranged from 72.7% for LIB to 86.4% for PSE. Good transferability was thus obtained within the Coffea genus. When focusing on the two species CAN and PSE, high genetic diversity, high polymorphic locus rates (above 80%) and a mean allele number per polymorphic locus of more than 3 were noted. The estimated null allele percentage was -11% for PSE and -9% for CAN. Sixty three percent (CAN) and 79.5% (PSE) of the fixation index (Fis) values were positive. The within-species polymorphism information content (PIC) distribution showed two modes for both species. Although the two species shared 30 polymorphic loci, no correlation between CAN and PSE PIC values was obtained. All of these data are discussed in relation to the polymorphism level and the potential use of these SSRs for subsequent analysis of genetic diversity or genetic mapping.

Inheritance of seed desiccation sensitivity in a coffee interspecific cross: evidence for polygenic determinism.

The genetic determinism of seed desiccation sensitivity was studied using a cross between two coffee species exhibiting a large difference for this trait, Coffea pseudozanguebariae (tolerant) and C. liberica (sensitive). Throughout the whole study, seed desiccation tolerance was quantified both in terms of water content and water activity. Whatever the parameter used, the level of seed desiccation tolerance in F1 hybrids corresponded to that of the mid-parent, thus indicating an additive inheritance of seed desiccation tolerance at the F1 level. A broad variation was observed among hybrids backcrossed to C. liberica (BCs) for seed desiccation tolerance, independent of the parameter used to quantify it. This variation was continuous and BCs showed transgression in the direction of the most desiccation sensitive parent, indicating (i) that desiccation tolerance is a polygenic trait in coffee species, and (ii) that C. pseudozanguebariae does not present the most favourable alleles for all the genes involved in seed desiccation tolerance. No significant difference was observed between the two reciprocal backcrosses, F1xC. liberica and C. libericaxF1, for the level of desiccation tolerance of their seeds, showing the absence of a maternal effect on this trait. There was no significant effect of the number of seeds harvested from each BC on the level of desiccation tolerance of its seeds. Moreover, there was no significant correlation within BCs between seed size, seed viability, and water content before desiccation and desiccation tolerance.

Chlorogenic acid content swap during fruit maturation in Coffea pseudozanguebariae: Qualitative comparison with leaves

Chlorogenic acids (CGA) are products of phenylpropanoid metabolism, i.e. one branch of the phenolic pathway. A wild species, Coffea pseudozanguebariae, native of East Africa, is a caffeine-free species with low CGA content (1.2% dmb in green beans). It is also used as a gene donor to improve C. canephora cup taste quality. In the current study, contents of the different CGA isomers were observed during the development in fruits and leaves. In both organs, CGA content decreased strongly during the growth and feruloylquinic acids (FQA) constituted most CGA. In fruits, a critical step was emphasised at the growth end, beyond which caffeoylquinic acid content (CQA) drastically increased. Previous results on beans suggest that the qualitative change concerned seeds and not pulp. The breeding implications and potential for further studies are discussed.

AFLP and SSR polymorphism in a Coffea interspecific backcross progeny [( C. heterocalyx x C. canephora) x C. canephora

An interspecific cross (BC 1) involving a species with one of the largest genomes in the Coffea genus [ Coffea heterocalyx (HET), qDNA = 1.74 pg] and a species with a medium-sized genome [ Coffea canephora (CAN), qDNA = 1.43 pg] was studied using two types of molecular markers, AFLP and SSR. One hundred and eighty eight AFLP bands and 34 SSR primer pairs were suitable for mapping. The total map length was 1,360 cM with 190 loci distributed in 15 linkage groups. The results were compared to those obtained previously on an interspecific BC 1 progeny involving a species with a medium-sized genome ( Coffea liberica var dewevrei, DEW) and a species with one of the smallest genomes ( Coffea pseudozanguebariae, PSE). They are discussed relative to three main points: (1) the relevance of the different marker types, (2) the genomic distribution of AFLP and SSR markers, and (3) the relation between AFLP polymorphism and genome size.

Genetic mapping of a caffeoyl-coenzyme A 3-O-methyltransferase gene in coffee trees. Impact on chlorogenic acid content.

Chlorogenic acids (CGA) are involved in the bitterness of coffee due to their decomposition in phenolic compounds during roasting. CGA mainly include caffeoyl-quinic acids (CQA), dicaffeoyl-quinic acids (diCQA) and feruloyl-quinic acids (FQA), while CQA and diCQA constitute CGA sensu stricto (CGA s.s.). In the two cultivated species Coffea canephora and Coffea arabica, CGA s.s. represents 88% and 95% of total CGA, respectively. Among all enzymes involved in CGA biosynthesis, caffeoyl-coenzyme A 3-O-methyltransferase (CCoAOMT) is not directly involved in the CGA s.s. pathway, but rather in an upstream branch leading to FQA through feruloyl-CoA. We describe how a partial cDNA corresponding to a CCoAOMT encoding gene was obtained and sequenced. Specific primers were designed and used for studying polymorphism and locating the corresponding gene on a genetic map obtained from an interspecific backcross between Coffea liberica var. Dewevrei and Coffea pseudozanguebariae. Offspring of this backcross were also evaluated for the chlorogenic acid content in their green beans. A 10% decrease was observed in backcross progenies that possess one C. pseudozanguebariae allele of the CCoAOMT gene. This suggests that CGA s.s. accumulation is dependent on the CCoAMT allele present and consequently on the activity of the encoded isoform, whereby CGA accumulation increases as the isoform activity decreases. Possible implications in coffee breeding are discussed.

Identification and mapping of a major gene (Ft1) involved in fructification time in the interspecific cross Coffea pseudozanguebariae x C. liberica var. Dewevrei: impact on caffeine content and seed weight.

Fructification time was studied in the interspecific cross Coffea pseudozanguebariae x C. liberica var. Dewevrei (PSE x DEW). Parental species, F(1) hybrids and offspring of the first backcross generation (BC(1)), consisting of F(1) x PSE (BCPSE) and F(1) x DEW (BCDEW) plants, were observed. Fructification time can be split into two independent visual phases: the full-growth period, from blooming up to the end of fruit growth, and the maturation phase, defined by the green to red color change. Fructification time was found to be an additive trait. The full-growth period showed a bimodal distribution in the BCDEW hybrid, suggesting the involvement of Ft1, a major gene that was mapped on linkage group E. The main effects of Ft1 were to lower caffeine content and 100-seed weight, without any impact on chlorogenic acid, trigonelline and sucrose contents. Two molecular markers were identified that bracket Ft1 and which could be used for early marker-assisted selection.

Trigonelline inheritance in the interspecific Coffea pseudozanguebariae×C. liberica var. dewevrei cross

Trigonelline alkaloid is present in coffee beans, and during roasting it gives rise to the major coffee aroma compounds (several alkyl-pyridines and pyrroles). In this study we investigated the genetic inheritance of trigonelline accumulation in green beans in an interspecific cross between a wild east African species, Coffea pseudozanguebariae (PSE) and the west African species C. liberica var. dewevrei (DEW). Trigonelline content was measured by HPLC in both parental species, F1 hybrids and the reciprocal backcross hybrids (BCDEW and BCPSE). The results showed that, on average, PSE accumulated twice as much trigonelline as DEW. No year effect or interaction (genotype×year) was recorded. Trigonelline showed high heritability (71%), which meant that the genotypic value could be easily estimated from the phenotypic value. However, the fact that this trait was not additive suggested the possibility of nucleo-cytoplasmic inheritance. This hypothesis was confirmed by: (1) similar levels of trigonelline content in the PSE, F1, BCPSE and BCDEW groups, all having the same maternal cytoplasm, and (2) the location of one nuclear QTL on the G linkage group.

Relations between and inheritance of chlorogenic acid contents in an interspecific cross between Coffea pseudozanguebariae and Coffea liberica var ‘dewevrei’

Chlorogenic acids (CGA) are phenolic compounds commonly found in green coffee beans. The main CGA classes are caffeoylquinic acids (CQA), dicaffeoylquinic acids (diCQA), and feruloylquinic acids (FQA). Each contains three isomers differing in the number and identity of the acylating residues. An interspecific cross between Coffea pseudozanguebariae (low CGA content) and C. liberica var ‘dewevrei’ (high CGA content) was investigated for CGA contents in F1 and back-cross hybrids. Relations within and between CGA classes were studied and confirmed the known biosynthesis pathway. A single major gene was noted for the 3-FQA isomer; absence was dominant. Additivity was found for most other isomers either with or without the transformation of variables. Conversely, most ratios were not additive, due to a curvilinear relation between some isomers. The consequences for breeding both in terms of cup taste improvement and disease resistance are discussed.

Levels of Cafestol, Kahweol, and Related Diterpenoids in Wild Species of the Coffee Plant Coffea

Coffee beans of commercially important Coffea species contain the diterpene cafestol, which raises serum cholesterol in humans. Coffee diterpenes also have shown anticarcinogenic properties in experimental animals. We determined levels of cafestol and other diterpenes in nine wild African Coffea species, in a search for a species without the cholesterol-raising cafestol. In addition, data on diterpene profiles might assist in the taxonomic grouping of Coffea species. Each Coffea species we investigated contained cafestol. Kahweol concentrations may be related to the geographical distribution of the Coffea species. 16-O-Methylcafestol was only present in Coffea canephora, Coffea stenophylla, and Coffea liberica var. dewevrei. A new diterpene was found in C. stenophylla and tentatively identified as 16-O-methylkahweol. Another four new diterpenes, which seem to contain an additional double bond compared to kahweol, were found in Coffea salvatrix and Coffea pseudozanguebariae, suggesting that these two species are closely related. In conclusion, cafestol is universally present in Coffea species. Amounts of kahweol, 16-O-methylcafestol, 16-O-methylkahweol, and the four newly found diterpenes present in various coffee beans may prove useful in the taxonomic classification of Coffea species. Keywords: Coffee diterpenes; African coffee beans; cholesterol; taxonomy